Endoscopic submucosal gel injection containing phycocyanin as well as preparation method and application of endoscopic submucosal gel injection

Abstract

The invention discloses an endoscopic submucosal gel injection containing phycocyanin as well as a preparation method and application of the endoscopic submucosal gel injection. Oxidized sodium alginate, carboxymethyl chitosan, calcium chloride and phycocyanin are utilized to construct a novel endoscopic submucosal gel injection for promoting wound healing, and the novel endoscopic submucosal gel injection is injected in a sol state; according to the present invention, the semi-solid gel is spontaneously formed at the injection site, is matched with the ESD surgical procedure, has the physical properties of injectability, long-time mucous membrane uplift, adhesion and the like, and further has the biological properties of anti-inflammation, anti-oxidation, safety and biodegradability, such that the endoscopic submucous injection can be performed, and the wound healing can be promoted;

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical materials, and in particular relates to an endoscopic submucosal gel injection containing phycocyanin, and a preparation method and application thereof. Background Art

[0002] Endoscopic submucosal dissection (ESD) is a minimally invasive technique that completely removes the diseased mucosa from the submucosal layer under endoscopy. Since it can completely remove early gastrointestinal tumors at one time under endoscopy, ESD has broad clinical application prospects. However, the cutting and peeling of the diseased mucosa during ESD is very difficult and requires high technical skills. The probability of complications such as perforation and bleeding is very high, which limits the widespread use of ESD.

[0003] The key to ESD surgery is to inject liquid into the submucosal layer to separate the mucosal layer and the muscular layer, thereby elevating the lesion, which helps to perform fast and safe endoscopic surgery. A variety of submucosal injection materials have been used in ESD, including normal saline and hypertonic saline, glucose solution, glycerol, hydroxypropyl methylcellulose, sodium hyaluronate, etc. However, these substances will diffuse rapidly during the operation, making it difficult to maintain a long-term mucosal augmentation effect. Repeated injections are required, which prolongs the operation time and may even cause tissue inflammatory reactions.

[0004] Hydrogels are composed of polymers with a large number of hydrophilic groups and have a three-dimensional network structure. Hydrogels have good biocompatibility and biodegradability, as well as adjustable mechanical and tissue adhesion properties. Their excellent physical and chemical properties have opened up many applications in the biomedical field, including wound healing, drug and cell delivery, cancer treatment, bioelectronics and tissue regeneration. One type is injectable hydrogels, which are mainly used in minimally invasive surgery in vivo. They are characterized by being liquid before and during injection and being able to quickly gel in situ after injection.

[0005] Submucosal injection materials are filled between the mucosal layer and the muscularis propria, which can lift the lesion and fully separate it from the muscularis propria, help improve the complete resection rate of the lesion, and reduce the occurrence of complications such as bleeding and perforation. Ideal submucosal injection materials should meet the following requirements: (1) They can be easily injected through an endoscopic needle with a length of about 2 m and an inner diameter of less than 2 mm; (2) The mucosal bulge can be maintained for a long time during surgery, and the mucosa does not collapse after incision; (3) They are degradable and do not cause local inflammation; (4) They have biological effects such as hemostasis and anti-inflammatory effects, and promote postoperative wound healing; (5) They improve the visibility of the intraoperative field of view.

[0006] However, some hydrogel-based submucosal injection materials have many defects. On the one hand, the photocrosslinking in situ curing of hydrogels requires ultraviolet irradiation, which may cause inflammation of residual tissue and is difficult to use clinically. On the other hand, the disposable endoscopic needles used clinically are usually 2.3m long. When thermosensitive hydrogels are delivered to the human body, they may reach the gel temperature and form local gel blocks, thereby blocking the needle. In addition, the cumbersome operation of the double-needle / two-step injection hydrogel system will cause unnecessary burden. Moreover, some hydrogel-based submucosal injection materials only have the physical effect of lifting the mucosa, but do not have the biological effect of promoting wound repair.

[0007] From the above analysis, it can be seen that the solution-based submucosal injections currently used in clinical practice are difficult to meet the needs of ESD, and some hydrogel-based submucosal injection materials have many defects and do not meet the application requirements of ESD. Therefore, there is an urgent need to develop an endoscopic submucosal gel injection that has both the physical ability to elevate lesions and the biological ability to repair wounds, so as to further improve the safety and effectiveness of ESD. Summary of the invention

[0008] The purpose of the present invention is to provide an endoscopic submucosal gel injection containing phycocyanin, a preparation method and application thereof, and a novel endoscopic submucosal gel injection for promoting wound healing is constructed by using oxidized sodium alginate, carboxymethyl chitosan, calcium chloride and phycocyanin. The endoscopic submucosal gel injection is injected in a sol state and spontaneously forms a semisolid gel at the injection site, which matches the ESD surgical procedure. At the same time, it has the physical properties of injectability, long-term mucosal protrusion, adhesion, etc., and also has the biological properties of anti-inflammatory, antioxidant and safe biodegradability. Therefore, it can not only perform endoscopic submucosal injection, but also promote wound healing.

[0009] The technical scheme of the present invention is: an endoscopic submucosal gel injection containing phycocyanin, comprising oxidized sodium alginate, carboxymethyl chitosan, calcium chloride and phycocyanin.

[0010] As a preferred technical solution, the endoscopic submucosal gel injection is prepared by mixing a mixed solution of carboxymethyl chitosan and phycocyanin, a calcium chloride solution, and an oxidized sodium alginate solution in sequence, wherein the concentration of carboxymethyl chitosan in the mixed solution of carboxymethyl chitosan and phycocyanin is 3% to 4% g / ml, and the concentration of phycocyanin is 0.875% to 1.75% g / ml; the concentration of calcium chloride in the calcium chloride solution is 0.5% to 1% g / ml; and the concentration of oxidized sodium alginate in the oxidized sodium alginate solution is 3% to 4% g / ml.

[0011] As a preferred technical solution, the volume ratio of the mixed solution of carboxymethyl chitosan and phycocyanin to the oxidized sodium alginate solution is 2:1.

[0012] As a preferred technical solution, the concentration of phycocyanin in the endoscopic submucosal gel injection is 0.5-1% g / ml.

[0013] A use of the endoscopic submucosal gel injection containing phycocyanin as described above in endoscopic submucosal dissection and postoperative wound healing.

[0014] A method for preparing the endoscopic submucosal gel injection containing phycocyanin as described above comprises the following steps:

[0015] Step S1: adding sodium alginate to ultrapure water to dissolve, then adding sodium periodate, stirring at a constant temperature and in the dark; after reacting for 6 to 8 hours, adding ethylene glycol to terminate the reaction; dialyzing the obtained mixed solution using a dialysis bag with a molecular weight cutoff of 3500 and freeze-drying to obtain sponge-like oxidized sodium alginate;

[0016] Step S2: dissolving the oxidized sodium alginate obtained in step S1 in ultrapure water to obtain an oxidized sodium alginate solution having an oxidized sodium alginate concentration of 3% to 4% g / ml;

[0017] Step S3: dissolving carboxymethyl chitosan and phycocyanin in ultrapure water in sequence to obtain a mixed solution of carboxymethyl chitosan and phycocyanin with a carboxymethyl chitosan concentration of 3% to 4% g / ml and a phycocyanin concentration of 0.875% to 1.75% g / ml;

[0018] Step S4: dissolving calcium chloride in ultrapure water to obtain a calcium chloride solution having a calcium chloride concentration of 0.5% to 1% g / ml;

[0019] Step S5: first add the calcium chloride solution to the mixed solution of carboxymethyl chitosan and phycocyanin, mix well, then add the oxidized sodium alginate solution, stir thoroughly until the pH is neutral, and obtain an endoscopic submucosal gel injection solution, wherein the concentration of phycocyanin is 0.5-1% g / ml.

[0020] As a preferred technical solution, the mass ratio of the sodium alginate to the sodium periodate in step S1 is 1:1.08.

[0021] As a preferred technical solution, the oxidation degree of the oxidized sodium alginate in step S1 is greater than 40%.

[0022] As a preferred technical solution, the volume ratio of the mixed solution of carboxymethyl chitosan and phycocyanin to the oxidized sodium alginate solution in step S5 is 2:1.

[0023] The advantages of the present invention are:

[0024] 1. The endoscopic submucosal gel injection containing phycocyanin of the present invention, and its preparation method and application, utilize oxidized sodium alginate, carboxymethyl chitosan, calcium chloride and phycocyanin to construct a new endoscopic submucosal gel injection for promoting wound healing. It is injected in a sol state and spontaneously forms a semisolid gel at the injection site, which matches the ESD surgical procedure. It also has the physical properties of injectability, long-term mucosal protrusion, adhesion, etc., and also has the biological properties of anti-inflammatory, antioxidant and safe biodegradability. Therefore, it can not only perform endoscopic submucosal injection, but also promote wound healing.

[0025] 2. The present invention utilizes oxidized sodium alginate, carboxymethyl chitosan, calcium chloride and phycocyanin to construct a novel endoscopic submucosal gel injection containing phycocyanin and promoting wound healing. It does not require the use of a corresponding cross-linking method with large uncertainty in the environment such as temperature and light, nor does it require step-by-step injection. It can be easily injected into the body after mixing in vitro using a suitable gelation time. At the same time, since the cross-linking of sodium alginate and calcium ions is fast and uneven, it is difficult to control the appropriate concentration. Therefore, the present invention adopts oxidized sodium alginate and carboxymethyl chitosan for cross-linking, rather than oxidized sodium alginate and calcium ions for cross-linking, and the cross-linking speed is uniform and controllable.

[0026] 3. In the preparation method of the endoscopic submucosal gel injection containing phycocyanin of the present invention, the endoscopic submucosal gel injection is prepared by mixing a mixed solution of carboxymethyl chitosan and phycocyanin, a calcium chloride solution, and an oxidized sodium alginate solution in sequence, wherein the volume ratio of the mixed solution of carboxymethyl chitosan and phycocyanin to the oxidized sodium alginate solution is 2:1, and the gelation speed is relatively slow at this time, which is convenient for adjusting the gelation time.

[0027] 4. The existing clinical use of sodium hyaluronate, glycerol fructose, hydroxyethyl starch and other solutions has a greater viscosity than normal saline. After injection, the diffusion to the surrounding tissues is slowed down, the tissue absorption is slowed down, and the effect of maintaining the height of the raised mucosa is better than normal saline. However, since it is essentially a flowable liquid, it still flows during cutting, and the ability to resist mucosal pressure is very poor, resulting in a rapid decrease in the height of the raised part, which does not fundamentally improve the problem. The present invention constructs a new type of endoscopic submucosal gel injection solution that promotes wound healing and contains phycocyanin, which makes the mucosal layer of the lesion bulge and separates it from the underlying muscle layer, improves the operating field and space, avoids injury to the muscle layer, reduces damage to blood vessels, and reduces the probability of occurrence. At the same time, the endoscopic submucosal gel injection solution containing phycocyanin can solidify in situ, will not diffuse to the surrounding tissues after injection, and will not flow out during the cutting process.

[0028] 5. The present invention constructs a novel endoscopic submucosal gel injection containing phycocyanin that promotes wound healing. Calcium ions are introduced on the basis of cross-linking of oxidized sodium alginate and carboxymethyl chitosan. The introduction of calcium ions will react with oxidized sodium alginate to form a double-network hydrogel. By controlling the amount of calcium ions, on the one hand, the gel can be added without increasing the injection resistance; on the other hand, it has stronger adhesion than the gel of only oxidized sodium alginate and carboxymethyl chitosan, and the degradation of the hydrogel is delayed, and it has a smaller swelling rate, which can provide guarantee for drug release.

[0029] 6. The present invention constructs a novel endoscopic submucosal gel injection containing phycocyanin that promotes wound healing and can increase tissue contrast under endoscopy. Methylene blue is usually added for staining in clinical practice, but no additional staining is required in the present invention; at the same time, the diffusion of methylene blue may cause blurred vision of the lesion during surgery, while phycocyanin can be better confined to the gel than methylene blue. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments will be briefly introduced below. The accompanying drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative work.

[0031] The present invention will be further described below in conjunction with the accompanying drawings and embodiments:

[0032] Figure 1 This is a comparison diagram of the relationship between the gel time and the concentration of CMCS and OSA solutions in Example 4;

[0033] Figure 2 This is a comparison diagram of the required injection resistance when V(OSA) / V(CMCS)=1 / 2, OSA concentration is 3% to 4% g / ml, and CMCS concentration is 3% to 4% g / ml in Example 5;

[0034] Figure 3 In this example 5, Ca is added 2+ Comparison chart of injection resistance required after injection;

[0035] Figure 4 In this example 5, Ca is added 2+ Comparison chart of gel time required after

[0036] Figure 5 HA sodium hyaluronate and OC2 / 1% PC, OC2 / 1% PC / Ca in Example 6 2+ Comparison of mucosal ridge height of hydrogel;

[0037] Figure 6HA sodium hyaluronate and OC2 / 1% PC, OC2 / 1% PC / Ca in Example 7 2+ 、OC2 / 0.07%MB / Ca 2+ 、OC2 / 0.5%PC / Ca 2+ Schematic diagram of the diffusion of hydrogels (arranged from left to right) within 2 h;

[0038] Figure 7 HA sodium hyaluronate and OC2 / 1% PC, OC2 / 1% PC / Ca in Example 7 2+ 、OC2 / 0.07%MB / Ca 2+ 、OC2 / 0.5%PC / Ca 2+ Comparison of hydrogel diffusion;

[0039] Figure 8 In this embodiment 8 (OC2 / PC, OC2 / PC / Ca 2+ ) Adhesion strength test result diagram;

[0040] Fig. 9 This is a graph showing the experimental results of Example 9 showing that phycocyanin can be used as an effective regulatory factor for macrophage M2 polarization;

[0041] Fig.10 In this embodiment 10 (HA sodium hyaluronate, OC2 / PC / Ca 2+ ) Maintain the height comparison of mucosal elevation;

[0042] Fig.11 This is a comparison diagram of the wound healing on the stomach in Example 10;

[0043] Fig.12 This is a comparison diagram of the gastric mucosal repair morphology under the pathological section HE display in this Example 10;

[0044] Fig.13 This is a comparison diagram of the repair morphology of the gastric mucosa in Example 10 shown by Sirius red staining. DETAILED DESCRIPTION

[0045] The above scheme is further described below in conjunction with specific examples. It should be understood that these examples are used to illustrate the present invention and are not limited to the scope of the present invention. The implementation conditions adopted in the examples can be further adjusted according to the conditions of the specific manufacturer, and the unspecified implementation conditions are usually the conditions in conventional experiments.

[0046] Example 1

[0047] Preparation of a basic hydrogel cross-linked with oxidized sodium alginate and carboxymethyl chitosan (OSA / CMCS hydrogel) comprises the following steps:

[0048] Dissolve 10 g of sodium alginate in 250 ml of ultrapure water, add 10.8 g of sodium periodate (mass of sodium alginate:mass of sodium periodate=1:1.08), stir at a constant temperature of about 25° C. in the dark, react for 6 to 8 hours, add 10 ml of ethylene glycol to terminate the reaction, dialyze the mixed solution for three days using a dialysis bag with a molecular weight cutoff of 3500, concentrate the liquid, and freeze-dry to obtain sponge-like oxidized sodium alginate (oxidation degree>40%).

[0049] Dissolve 0.3, 0.4, and 0.5 g of oxidized sodium alginate and carboxymethyl chitosan in 10 ml of ultrapure water to form oxidized sodium alginate solutions and carboxymethyl chitosan solutions with concentrations of 3% g / ml, 4% g / ml, and 5% g / ml, respectively, and control the pH to be neutral.

[0050] Oxidized sodium alginate solution and carboxymethyl chitosan solution of different concentrations were mixed in different volume ratios (1:2, 1:1, 2:1) according to an orthogonal design table to form a basic hydrogel cross-linked by oxidized sodium alginate and carboxymethyl chitosan (OSA / CMCS hydrogel).

[0051] Example 2

[0052] Preparation of Ca 2+ The basic hydrogel (OSA / CMCS / Ca 2+ hydrogel), comprising the steps of:

[0053] Dissolve 10 g of sodium alginate in 250 ml of ultrapure water, add 10.8 g of sodium periodate (mass of sodium alginate:mass of sodium periodate=1:1.08), stir at a constant temperature of about 25° C. in the dark, react for 6 to 8 hours, add 10 ml of ethylene glycol to terminate the reaction, dialyze the mixed solution for three days using a dialysis bag with a molecular weight cutoff of 3500, concentrate the liquid, and freeze-dry to obtain sponge-like oxidized sodium alginate (oxidation degree>40%).

[0054] 0.4 g of oxidized sodium alginate was dissolved in 10 ml of ultrapure water to form oxidized sodium alginate solutions with a concentration of 4% g / ml, and the pH was controlled to be neutral.

[0055] 0.3 g of carboxymethyl chitosan was dissolved in 10 ml of ultrapure water to form a carboxymethyl chitosan solution with a concentration of 3% g / ml, and the pH was controlled to be neutral.

[0056] 0.1 g of calcium chloride was dissolved in 10 ml of ultrapure water to form a calcium chloride solution with a calcium chloride concentration of 1% g / ml.

[0057] Take 1 ml of 3% g / ml carboxymethyl chitosan solution, add 0.25 ml of 1% g / ml calcium chloride solution, mix well, then add 0.5 ml of 4% g / ml sodium alginate solution to form a hydrogel (OC2 / Ca 2+ hydrogel).

[0058] Example 3

[0059] Preparation of endoscopic submucosal gel injection containing phycocyanin (OSA / CMCS / PC / Ca 2+ A method for preparing a hydrogel comprises the following steps:

[0060] First, 10 g of sodium alginate was dissolved in 250 ml of ultrapure water, and then 10.8 g of sodium periodate (wherein the mass of sodium alginate: the mass of sodium periodate = 1:1.08) was added, and the mixture was stirred at a constant temperature of about 25° C. in the dark. After reacting for 6 to 8 hours, 10 ml of ethylene glycol was added to terminate the reaction. The obtained mixed solution was dialyzed for three days using a dialysis bag with a molecular weight cutoff of 3500, and the liquid was concentrated and freeze-dried to obtain sponge-like oxidized sodium alginate (with an oxidation degree of >40%).

[0061] 0.4 g of oxidized sodium alginate was dissolved in 10 ml of ultrapure water to form a solution with an oxidized sodium alginate concentration of 4% g / ml, and the pH was controlled to be neutral.

[0062] 0.3 g of carboxymethyl chitosan was dissolved in 10 ml of ultrapure water to form a solution with a carboxymethyl chitosan concentration of 3% g / ml, the pH was controlled to be neutral, and phycocyanin was added to obtain a mixed solution of carboxymethyl chitosan and phycocyanin with phycocyanin concentrations of 0.875% g / ml and 1.75% g / ml.

[0063] 0.1 g of calcium chloride was dissolved in 10 ml of ultrapure water to form a calcium chloride solution with a calcium chloride concentration of 1% g / ml.

[0064] Take 1 ml of a mixed solution of carboxymethyl chitosan and phycocyanin containing high concentration (1.75% g / ml) phycocyanin, add 0.25 ml of 1% g / ml calcium chloride solution, mix well, and then add 0.5 ml of 4% g / ml oxidized sodium alginate solution to form an endoscopic submucosal gel injection containing phycocyanin (OC2 / 1% PC / Ca 2+ The concentration of phycocyanin in the endoscopic submucosal gel injection is 1% g / ml.

[0065] Take 1 ml of the mixed solution of carboxymethyl chitosan and phycocyanin containing low concentration (0.875% g / ml), add 0.25 ml of 1% g / ml calcium chloride solution, mix well, and then add 0.5 ml of 4% g / ml oxidized sodium alginate solution to form an endoscopic submucosal gel injection containing phycocyanin (OC2 / 0.5% PC / Ca 2+ The concentration of phycocyanin in the endoscopic submucosal gel injection is 0.5% g / ml.

[0066] Example 4

[0067] The gel time of the basic hydrogel (OSA / CMCS hydrogel) cross-linked with oxidized sodium alginate and carboxymethyl chitosan prepared in Example 1 was measured using the inversion method, and the steps were as follows:

[0068] First, the oxidized sodium alginate solution and carboxymethyl chitosan solution of different concentrations prepared in Example 1 were placed in a 37° C. incubator for 30 min, and the solution temperature was controlled at 37° C. The solutions were added to a small centrifuge tube in sequence, followed by a magnet, and then placed on a magnetic stirrer, and the tube was flipped every 10 seconds until a hydrogel was formed at the bottom of the ep tube and did not fall during the flipping process, and the final gelation time was recorded.

[0069] Table 1: Factor level table

[0070]

[0071] Table 2: Orthogonal design table

[0072]

[0073] According to Table 1 and Table 2, the orthogonal experimental analysis shows that the range R(C)>R(A)>R(B), and it can be obtained that the volume ratio of the two solutions has a greater impact on the gel time. The relationship between the gel time and the volume ratio of the solution: V(OSA) / V(CMCS)==1 / 2, the gel time is longer; V(OSA) / V(CMCS)=1 / 1, the gel time is moderate; V(OSA) / V(CMCS)=2 / 1, the gel time is shorter.

[0074] The relationship between the gel time and the concentration of the two solutions (V(OSA) / V(CMCS)=1 / 1, the gel time is easy to measure) was studied, and the results are as follows Figure 1 When the volume ratio and OSA concentration are constant, the greater the CMCS concentration, the longer the gel time; when the volume ratio and CMCS concentration are constant, the greater the OSA concentration, the shorter the gel time.

[0075] Example 5

[0076] Design a device to measure injection resistance, use a 1.5m long, 2mm diameter infusion extension tube (with the filter membrane cut off) + a 20G needle, and connect it to a 10ml syringe.

[0077] In the oxidized sodium alginate solution and carboxymethyl chitosan solution prepared in Example 1, 2 ml of oxidized sodium alginate solution of different concentrations and 2 ml or 4 ml of carboxymethyl chitosan solution of different concentrations were taken, mixed and put into a syringe, and a 1 kg weight was added to the syringe.

[0078] Under the gravity of the weight, the liquid in the syringe flows out through the infusion tube and is collected by the EP tube. The injection volume is recorded at 1 minute, 2 minutes, and 3 minutes respectively.

[0079] When the volume ratio of oxidized sodium alginate solution to carboxymethyl chitosan solution is 1 / 1 or 2 / 1, it is difficult to inject. When the volume ratio is 1 / 2, it can be injected. When the concentration of oxidized sodium alginate solution is 3-4%, the concentration of carboxymethyl chitosan solution is 3-4%, it can be injected. In summary, we choose V(OSA) / V(CMCS)=1 / 2, OSA concentration is 3%-4% g / ml, CMCS concentration is 3%-4% g / ml for the next step of the experiment. The results are as follows Figure 2 .

[0080] The injection resistance was small when the concentration of oxidized sodium alginate solution was 3-4% and the concentration of carboxymethyl chitosan solution was 3%. The hydrogel formed when the concentration of oxidized sodium alginate solution was 3%, the concentration of carboxymethyl chitosan solution was 3%, and V(OSA) / V(CMCS)=1 / 2 was named OC1; the hydrogel formed when the concentration of oxidized sodium alginate solution was 4%, the concentration of carboxymethyl chitosan solution was 3%, and V(OSA) / V(CMCS)=1 / 2 was named OC2. OC1 had a longer gel time and weaker cross-linking strength, so we chose OC2 for the next experiment.

[0081] According to Examples 2 and 3, Ca was added 2+ and phycocyanin, and the injection resistance was measured. Figure 3 , 4 The results showed that the addition of Ca 2+ , has almost no effect on injection resistance, but will accelerate gelation. Adding phycocyanin will increase injection resistance to a certain extent.

[0082] Example 6

[0083] In vitro, 0.4% g / ml HA sodium hyaluronate (0.4% HA / 0.07% MB, a submucosal injection material used clinically) and OC2 / 1% PC, OC2 / 1% PC / Ca 2+The hydrogels were injected into the submucosal layer of the pig stomach to evaluate their submucosal lifting performance. The changes in submucosal height of each group within 2.5 h were measured with a vernier caliper. Figure 5 , it can be observed that the hydrogel can still maintain more than 90% of the mucosal elevation height after 2.5 h, while the sodium hyaluronate group rapidly decreased after 0.5 h.

[0084] Example 7

[0085] The materials were injected into the submucosal layer of pig stomach in vitro. After 2 hours, the submucosal layer was cut open to observe the diffusion. Figure 6 From left to right: 0.4% g / ml HA sodium hyaluronate containing 0.07% methylene blue (MB) (the submucosal injection material used clinically 0.4% HA / 0.07% MB) and OC2 / 1% PC, OC2 / 1% PC / Ca 2+ 、OC2 / 0.07%MB / Ca 2+ 、OC2 / 0.5%PC / Ca 2+ Hydrogel. Inject the above materials into water, let it stand, and observe the diffusion of phycocyanin and methylene blue within 2 hours. Figure 7 , the results show that methylene blue diffuses faster in tissue and water, which may cause unclear vision of lesions during surgery.

[0086] Example 8

[0087] The evaluation was performed using a universal test device with a speed of 5 mm / min. A pig skin tissue block with a size of 5.0 cm*1.5 cm was immersed in simulated gastric fluid at 37°C for 1.5 h to simulate the in vivo environment. Subsequently, 50 μL (OC2 / PC, OC2 / PC / Ca 2+ ) The hydrogel was applied to the contact area (1.0 cm×1.5 cm) where two wet pig skin tissues were connected. Adhesion strength (Pa=N / m 2 ) is calculated using the equation. Figure 8 ,The results showed that the hydrogel had a certain degree of adhesion and could remain on the wound of ESD surgery for a period of time.

[0088] Example 9

[0089] RAW264.7 cells (density 5×10 5 ) were cultured in six-well plates. 300ng / ml LPS induced M1 polarization of cells. 400, 200, and 100ug / ml phycocyanin were added to the three experimental groups at the same time. OC2 / PC / Ca2+ with PC content of 1%, 0.5%, and 0% were added to the three experimental groups at the same time. 2+The hydrogel extract was co-cultured for 24 h. The samples were treated with antibodies CD11b-FITC, CD86-PE-Cy, and CD206-APC. Each sample was detected by flow cytometry. Fig. 9 The results showed that phycocyanin could significantly increase the ratio of CD206 / CD86 (M2 / M1), indicating that phycocyanin and hydrogels containing phycocyanin can serve as effective regulators of macrophage M2 polarization.

[0090] Example 10

[0091] Pigs were used as experimental animal models, and endoscopes, high-frequency electrosurgical units, endoscopic mucosal injection needles, operating rooms, and respiratory and heart rate monitors were prepared. Before surgery, the pigs were fasted for 48 hours, and laxatives were given 24 hours before surgery. They were anesthetized before surgery. ESD surgery: Two sites were selected for submucosal injection in the gastric antrum, 0.4% sodium hyaluronate was used as the control, and the experimental group was OC2 / 1% PC / Ca 2+ . Obtain images of mucosal bulges within 60 minutes. Use an electric knife to cut the surrounding mucosa along the outer edge and all the mucosa under the mucosa. Feed liquid food, supplement antibiotics and proton pump inhibitors for one week after surgery. Then feed a normal diet until euthanasia on the 14th day after surgery, and collect tissues. Fig.10 The hydrogel of the present invention can better maintain the height of the mucosal bulge during surgery. The pigs were killed on the 14th day, and the wound healing on the stomach was as follows: Fig.11 The left side is the hydrogel experimental group of the present invention, and the right side is the sodium hyaluronate control group. It can be seen that the mucosal defect in the experimental group is smaller than that in the control group. HE and Sirius red staining of pathological sections show that Fig.12 , 13 The gastric mucosal repair morphology of the gel group of the present invention is better and closer to that of the normal group, and the collagen fiber content is higher.

[0092] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. An endoscopic submucosal gel injection containing phycocyanin, characterized in that: Includes oxidized sodium alginate, carboxymethyl chitosan, calcium chloride and phycocyanin.

2. The endoscopic submucosal gel injection containing phycocyanin according to claim 1, characterized in that: The endoscopic submucosal gel injection is prepared by sequentially mixing a carboxymethyl chitosan and phycocyanin mixed solution, a calcium chloride solution, and an oxidized sodium alginate solution, wherein the concentration of carboxymethyl chitosan in the carboxymethyl chitosan and phycocyanin mixed solution is 3% to 4% g / ml, and the concentration of phycocyanin is 0.875% to 1.75% g / ml; the concentration of calcium chloride in the calcium chloride solution is 0.5% to 1% g / ml; and the concentration of oxidized sodium alginate in the oxidized sodium alginate solution is 3% to 4% g / ml.

3. The endoscopic submucosal gel injection containing phycocyanin according to claim 2, characterized in that: The volume ratio of the carboxymethyl chitosan and phycocyanin mixed solution to the oxidized sodium alginate solution is 2:

1.

4. The endoscopic submucosal gel injection containing phycocyanin according to claim 1, characterized in that: The concentration of phycocyanin in the endoscopic submucosal gel injection is 0.5-1% g / ml.

5. Use of the endoscopic submucosal gel injection containing phycocyanin as claimed in any one of claims 1 to 4 in endoscopic submucosal dissection and postoperative wound healing.

6. A method for preparing the endoscopic submucosal gel injection containing phycocyanin according to any one of claims 1 to 4, characterized in that: The steps include: Step S1: adding sodium alginate to ultrapure water to dissolve, then adding sodium periodate, stirring at a constant temperature and in the dark; after reacting for 6 to 8 hours, adding ethylene glycol to terminate the reaction; dialyzing the obtained mixed solution using a dialysis bag with a molecular weight cutoff of 3500 and freeze-drying to obtain sponge-like oxidized sodium alginate; Step S2: dissolving the oxidized sodium alginate obtained in step S1 in ultrapure water to obtain an oxidized sodium alginate solution having an oxidized sodium alginate concentration of 3% to 4% g / ml; Step S3: dissolving carboxymethyl chitosan and phycocyanin in ultrapure water in sequence to obtain a mixed solution of carboxymethyl chitosan and phycocyanin with a carboxymethyl chitosan concentration of 3% to 4% g / ml and a phycocyanin concentration of 0.875% to 1.75% g / ml; Step S4: dissolving calcium chloride in ultrapure water to obtain a calcium chloride solution having a calcium chloride concentration of 0.5% to 1% g / ml; Step S5: first add the calcium chloride solution to the mixed solution of carboxymethyl chitosan and phycocyanin, mix well, then add the oxidized sodium alginate solution, stir thoroughly until the pH is neutral, and obtain an endoscopic submucosal gel injection solution, wherein the concentration of phycocyanin is 0.5-1% g / ml.

7. The method for preparing the endoscopic submucosal gel injection containing phycocyanin according to claim 6, characterized in that: The mass ratio of the sodium alginate to the sodium periodate in step S1 is 1:1.

08.

8. The method for preparing the endoscopic submucosal gel injection containing phycocyanin according to claim 6, characterized in that: The oxidation degree of the oxidized sodium alginate in step S1 is greater than 40%.

9. The method for preparing the endoscopic submucosal gel injection containing phycocyanin according to claim 6, characterized in that: In step S5, the volume ratio of the mixed solution of carboxymethyl chitosan and phycocyanin to the oxidized sodium alginate solution is 2:1.

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